Peptide:
N-glycosidase F (PNGase F) is an asparagine amidase produced by Flavobacterium
meningosept-icum that serves as a useful tool in the research on protein
N-glycosylation. The cleavage site of PNGase F is the amide bond between
N-acetylglucosamine (GlcNAc) and aspartate residues on the medial side of the
glycoprotein, and converts aspartyl to aspartic acid on the enzymolysis
protein. This productoverexpressed in E.coli
and used for complete deglycosylation of antibodies and their associated
proteins
Storage
Solution : 40U/ul PNGase
F、20 mM Tris-HCl、50 mM NaCl, 5 mM EDTA
(pH 7.5 @ 25°C)
10*NP-40: 10% NP-40 in
MilliQ-H2O
10*Denaturing Buffer: 5% SDS、400 mM DTT
10*Reaction Buffer: 500mM
Tris-HCl (pH 7.5 @ 25°C)
一. Denaturing Reaction Conditions:
1. Combine 1-20 µg of glycoprotein, 1 µl of Denaturing Buffer (10X) and H2O (if necessary) to make a 10 µl total reaction volume.
2. Denaturation is terminated by heating to 100℃ for 10-20min and cooling to room temperature.
3. Make a total reaction volume of 20 µl by adding 2 µl Reaction Buffer (10×), 2 µl 10% NP-40 and 6 µl H2O.
4. Add 1 µl PNGase F, mix gently.
5. Enzymatic digestion at 37℃ for 1h
6. Analyze by the method of SDS-PAGE
二. Non-Denaturing Reaction Conditions:
1. Combine 1-20 µg of glycoprotein, 2 µl of Reaction Buffer (10×) and H2O (if necessary) to make a 20 µl total reaction volume.
2. Add 2-5 µl PNGase F, mix gently.
3. Enzymatic digestion at 37°C for 4 - 24 hours.
4. Analyze by the method of SDS-PAGE
One unit of enzyme activity refers to the amount of enzyme required to remove more than 95% of carbohydrate from 10μg denatured RNaseB at 37℃ for 1 hour in a 10μL reaction system.
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